The principal goal of our research program has been to characterize the dynamic state of biological membranes, with the objective of exploring the causal relationships between molecular interactions, membrane protein mobilities, and specific membrane functions. To understand the function of cell membranes, one must understand the constraints on the motion of membrane molecules and how cells maintain specialized functional regions on the cell surface. In this proposal, we focus on three aspects of the plasma membrane: factors that restrict the mobility of membrane proteins, diffusion barriers at the boundaries of surface domains, and directed lateral movement of proteins on the cell surface. To better understand factors that control the mobility of membrane proteins, we will combine the techniques of fluorescence redistribution after photobleaching (FRAP), biochemistry, and molecular biology to investigate extracellular constraints to diffusion of glycosylphosphatidylinositol (GPI)-anchored proteins on guinea pig sperm. We will also measure diffusion of the beta-adrenergic receptor and its interacting G protein, Gs, to assess whether either one or both components are diffusing in the membrane, as proposed by the floating receptor hypothesis, and to examine how diffusion of these components affects their function in signal transduction. The localization of membrane components creates specific functional domains on the cell surface. We have demonstrated that barriers to membrane protein diffusion in the sperm plasma membrane retain freely diffusing proteins in specific domains. Digital imaging techniques and FRAP will be used to investigate how barriers function in both the maintenance and biogenesis of sperm plasma membrane domains, and mAbs will be used to identify components of the barriers. We will also explore whether membrane components of the G protein signal transduction pathway are localized in polarized epithelial cells, and if so, determine if diffusion barriers are involved in localizing these components. One mechanism that cells use to regulate the spatial organization of membrane proteins is directed lateral protein movement in the membrane. We will investigate two cases of specific, lateral migrations of the PH-20 protein (a GPI-anchored protein) of guinea pig sperm. The proposed studies will extend previous evidence that one case of PH-20 protein migration involves active translocation, and will characterize extracellular elements that may serve to translocate PH-20 protein during the migration. We will also continue to develop new methods of studying membrane dynamics. We will develop means to identify and account for factors that affect the measurement of diffusion coefficients by FRAP, including deviations from single-exponential photobleaching kinetics and diffusion during bleaching. We also introduce two new methods to study membrane dynamics. The first is a theoretical model that allows the use of multipoint analysis of FRAP to obtain quantitative information on both domain-like structure in membranes and possible fluorophore-sensitized membrane photodamage, and compares them to motions of colloidal gold particles. The second is a technique that uses fluorescence photobleaching anisotrophy to measure restrictions to angular mobility of membrane components.